DNA encoding insecticidal CRY9Fa Bacillus thuringiensis proteins and recombinant hosts expressing same

ABSTRACT

The present invention relates to new DNA sequences encoding an insecticidal Cry9Fa protein and insecticidal parts thereof, which are useful to protect plants from insect damage. Also included herein are micro-organisms and plants transformed with a DNA sequence encoding an insecticidal Cry9Fa protein and processes for controlling insects and to obtain a plant resistant to insects.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/600,831, filed Nov. 17, 2006, which is a divisional of U.S.application Ser. No: 10/614,524, filed Jul. 8, 2003, which is acontinuation of U.S. application Ser. No: 09/739,243, filed Dec. 19,2000, which claims the benefit of U.S. Ser. No. 60/173,387, filed Dec.28, 1999, each of which is incorporated herein by reference in itsentirety for all purposes.

INTRODUCTION

The present invention relates to new DNA sequences encoding insecticidalproteins produced by Bacillus thuringiensis strains. Particularly, newDNA sequences encoding proteins designated as Cry9Fa, Cry1Jd, and Cry1Bfare provided which are useful to protect plants from insect damage. Alsoincluded herein are micro-organisms and plants transformed with at leastone of the newly isolated genes so that they are useful to confer insectresistance by expression of insecticidal protein.

BACKGROUND OF THE INVENTION

(i) Field of the Invention

Bt or Bacillus thuringiensis is well known for its specific toxicity toinsect pests, and has been used since almost a century to control insectpests of plants. In more recent years, transgenic plants expressing Btproteins were made which were found to successfully control insectdamage on plants (e.g., Vaeck et al., 1987).

Despite the isolation of a number of Bt crystal protein genes, thesearch for new genes encoding insecticidal proteins continues. Indeed,insecticidal Bt crystal proteins are known to have a relatively narrowtarget insect range compared to chemical insecticides. Also, havingmultiple toxins active on the same target insect species allows the useof proteins having different modes of action so that insect resistancedevelopment can be prevented or delayed.

(ii) Description of Related Art

Previously, several types of Cry1B-, Cry1J-, and Cry9-proteins wereidentified (see Crickmore et al., 1998, incorporated herein byreference, for all details).

The new Cry1Bf protein has the closest sequence identity with the Cry1Beprotein (Payne et al, 1998, U.S. Pat. No. 5,723,758), but still differsin about 14 percent of the amino acid sequence of its toxic proteinfragment with the toxic fragment of the Cry1Be protein. The closestsequence identity with the Cry1Jd toxic fragment was found in the toxicfragment of the Cry1Jc1 protein (U.S. Pat. No. 5,723,758), but the toxicfragments of both proteins still differ in about 18% of their amino acidsequence.

The closest sequence identity with the Cry9Fa toxic fragment was foundwith the toxic fragment of the Cry9Ea1protein as described by Midoh etal. (PCT Patent publication WO 98/26073) and Narva et al. (PCT patentpublication WO 98/00546), but the toxic fragments of the Cry9Fa andCry9Ea proteins still differ in about 21% of their amino acid sequence.

SUMMARY OF THE INVENTION

In accordance with this invention is provided a DNA sequence encoding aprotein comprising the amino acid sequence selected from the groupconsisting of: a) the amino acid sequence of the insecticidaltrypsin-digestion fragment of the protein encoded by the cry1Bf genedeposited at the BCCM-LMBP under accession number LMBP 3986, b) theamino acid sequence of the insecticidal trypsin-digestion fragment ofthe protein encoded by the cry1Jd gene deposited at the BCCM-LMBP underaccession number LMBP 3983, and c) the amino acid sequence of theinsecticidal trypsin-digestion fragment of the protein encoded by thecry9Fa gene deposited at the BCCM-LMBP under accession number LMBP 3984.

Particularly preferred in accordance with this invention is a DNAsequence encoding a protein comprising the amino acid sequence selectedfrom the group consisting of: the amino acid sequence of an insecticidalfragment of the protein of SEQ ID No. 2, the amino acid sequence of aninsecticidal fragment of the protein of SEQ ID No. 4, and the amino acidsequence of an insecticidal fragment of the protein of SEQ ID No. 6;alternatively, a DNA encoding a protein comprising the amino acidsequence of the group selected from: the amino acid sequence of SEQ IDNo. 2, the amino acid sequence of SEQ ID No. 4, the amino acid sequenceof SEQ ID No. 6; or a DNA sequence comprising the DNA sequence of SEQ IDNo. 1, SEQ ID No. 3, or SEQ ID No. 5.

Further, in accordance with this invention are provided DNA sequencesencoding at least the following portions of the newly-isolated proteins:the amino acid sequence of SEQ ID No. 2 from amino acid position 1 toamino acid position 640, the amino acid sequence of SEQ ID No. 4 fromamino acid position 1 to amino acid position 596, and the amino acidsequence of SEQ ID No. 6 from amino acid position 1 to amino acidposition 652.

Further, in accordance with this invention are provided the above DNAsequences comprising an artificial DNA sequence having a different codonusage compared to the naturally occurring DNA sequence but encoding thesame protein or its insecticidal fragment.

Even further provided in accordance with this invention is a proteincomprising the amino acid sequence selected from the group consistingof: a) the amino acid sequence of the insecticidal trypsin-digestionfragment of the protein encoded by the cry1Bf gene deposited at theBCCM-LMBP under accession number LMBP 3986, b) the amino acid sequenceof the insecticidal trypsin-digestion fragment of the protein encoded bythe cry1Jd gene deposited at the BCCM-LMBP under accession number LMBP3983, and c) the amino acid sequence of the insecticidaltrypsin-digestion fragment of the protein encoded by the cry9Fa genedeposited at the BCCM-LMBP under accession number LMBP 3984.

Particularly preferred herein is a protein comprising the amino acidsequence selected from the group consisting of: the amino acid sequenceof an insecticidal fragment of the protein of SEQ ID No. 2, the aminoacid sequence of an insecticidal fragment of the protein of SEQ ID No.4, and the amino acid sequence of an insecticidal fragment of theprotein of SEQ ID No. 6; alternatively a protein, comprising the aminoacid sequence selected from the group consisting of: the amino acidsequence of SEQ ID No. 2 from amino acid position 1 to amino acidposition 640, the amino acid sequence of SEQ ID No. 4 from amino acidposition 1 to amino acid position 596, and the amino acid sequence ofSEQ ID No. 6 from amino acid position 1 to amino acid position 652; or aprotein comprising the amino acid sequence of SEQ ID No. 2, SEQ ID No.4, or SEQ ID No. 6.

Also provided herein are chimeric genes comprising the DNA as definedabove under the control of a plant-expressible promoter, and plantcells, plants or seeds transformed to contain those chimeric genes,particularly plant cells, plants, or seeds selected from the groupconsisting of: corn, cotton, rice, oilseed rape, Brassica species,eggplant, soybean, potato, sunflower, tomato, sugarcane, tea, beans,tobacco, strawberry, clover, cucumber, watermelon, pepper, oat, barley,wheat, dahlia, gladiolus, chrysanthemum, sugarbeet, sorghum, alfalfa,and peanut. In accordance with this invention, the chimeric gene can beintegrated in the nuclear or chloroplast DNA of the plant cells.

Further in accordance with this invention are provided micro-organisms,transformed to contain any of the above DNA sequences, particularlythose selected from the genus Agrobacterium, Escherichia, or Bacillus.

Also provided herein is a process for controlling insects, comprisingexpressing any of the above DNA sequences in a host cell, particularlyplant cells, and contacting insects with said host cells, and a processfor rendering a plant resistant to insects, comprising transformingplants cells with any of the above DNA sequences or chimeric genes, andregenerating transformed plants from such cells which are resistant toinsects.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In accordance with this invention, DNA sequences encoding new Bt toxinshave been isolated and characterized. The new genes were designatedcry1Bf, cry1Jd and cry9Fa, and their encoded proteins Cry1Bf, Cry1Jd andCry9Fa.

In accordance with this invention “Cry1Bf protein” refers to any proteincomprising the smallest protein fragment of the amino acid sequence ofSEQ ID No. 2 which retains insecticidal activity, particularly anyprotein comprising the amino acid sequence from the amino acid atposition 1 to the amino acid at position 640 in SEQ ID No. 2, includingbut not limited to the complete protein with the amino acid sequence ofSEQ ID No. 2. This includes hybrids or chimeric proteins comprising thesmallest toxic protein fragment, as well as proteins containing at leastone of the three functional domains of the toxic fragment of SEQ ID No.2. The term “DNA/protein comprising the sequence X”, as used herein,refers to a DNA or protein including or containing at least the sequenceX, so that other nucleotide or amino acid sequences can be included atthe 5′ (or N-terminal) and/or 3′ (or C-terminal) end, e.g. (thenucleotide sequence of) a selectable marker protein as disclosed in EP 0193 259.

In accordance with this invention, “Cry9Fa protein” or “Cry9F protein”refers to any protein comprising the smallest protein fragment of theamino acid sequence of SEQ ID No. 6 which retains insecticidal activity,particularly any protein comprising the amino acid sequence from theamino acid at position 1 to the amino acid at position 652 in SEQ ID No.6, including but not limited to the complete protein with the amino acidsequence of SEQ ID No. 6. This includes hybrids or chimeric proteinscomprising the smallest toxic protein fragment, as well as proteinscontaining at least one of the three functional domains of the toxicfragment of SEQ ID No. 6.

In accordance with this invention, “Cry1Jd protein” refers to anyprotein comprising the smallest protein fragment of the amino acidsequence of SEQ ID No. 4 which retains insecticidal activity,particularly any protein comprising the amino acid sequence from theamino acid at position 1 to the amino acid at position 596 in SEQ ID No.4, including but not limited to the complete protein with the amino acidsequence of SEQ ID No. 4. This includes hybrids or chimeric proteinscomprising the smallest toxic protein fragment, as well as proteinscontaining at least one of the three functional domains of the toxicfragment of SEQ ID No. 4.

As used herein, the terms “cry1Bf DNA”, “cry9Fa DNA”, or “cry1Jd DNA”,refer to any DNA sequence encoding the Cry1Bf, Cry9Fa, or Cry1Jdprotein, respectively, as defined above. This includes naturallyoccurring, artificial or synthetic DNA sequences encoding the newlyisolated proteins or their insecticidal fragments as defined above. Alsoincluded herein are DNA sequences encoding insecticidal proteins whichare similar enough to the coding regions of the genomic DNA sequencesdeposited or the sequences provided in the sequence listing so that theycan (i.e., have the ability to) hybridize to these DNA sequences understringent hybridization conditions. Stringent hybridization conditions,as used herein, refers particularly to the following conditions:immobilizing the relevant genomic DNA sequences on a filter, andprehybridizing the filters for either 1 to 2 hours in 50% formamide, 5%SSPE, 2×Denhardt's reagent and 0.1% SDS at 42° C. or 1 to 2 hours in6×SSC, 2×Denhardt's reagent and 0.1% SDS at 68° C. The denatured labeledprobe is then added directly to the prehybridization fluid andincubation is carried out for 16 to 24 hours at the appropriatetemperature mentioned above. After incubation, the filters are thenwashed for 20 minutes at room temperature in 1×SSC, 0.1% SDS, followedby three washes of 20 minutes each at 68° C. in 0.2×SSC and 0.1% SDS. Anautoradiograph is established by exposing the filters for 24 to 48 hoursto X-ray film (Kodak XAR-2 or equivalent) at −70° C. with anintensifying screen. Of course, equivalent conditions and parameters canbe used in this process while still retaining the desired stringenthybridization conditions. One of such equivalent conditions includes:immobilizing the relevant genomic DNA sequences on a filter, andprehybridizing the filters for either 1 to 2 hours in 50% formamide, 5%SSPE, 2×Denhardt's reagent and 0.1% SDS at 42° C. or 1 to 2 hours in6×SSC, 2×Denhardt's reagent and 0.1% SDS at 68° C. The denatured (dig-or radio-)labeled probe is then added directly to the prehybridizationfluid and incubation is carried out for 16 to 24 hours at theappropriate temperature mentioned above. After incubation, the filtersare then washed for 30 minutes at room temperature in 2×SSC, 0.1% SDS,followed by 2 washes of 30 minutes each at 68° C. in 0.5×SSC and 0.1%SDS. An autoradiograph is established by exposing the filters for 24 to48 hours to X-ray film (Kodak XAR-2 or equivalent) at −70° C. with anintensifying screen

“Insecticidal activity” of a protein, as used herein, means the capacityof a protein to kill insects when such protein is fed to insects,preferably by expression in a recombinant host such as a plant“Insect-controlling amounts” of a protein, as used herein, refers to anamount of protein which is sufficient to limit damage on a plant byinsects feeding on such plant to commercially acceptable levels, e.g. bykilling the insects or by inhibiting the insect development or growth insuch a manner that they provide less damage to a plant and plant yieldis not significantly adversely affected.

In accordance with this invention, insects susceptible to the new Cryproteins of the invention are contacted with this protein ininsect-controlling amounts, preferably insecticidal amounts. “Cryprotein” or “Cry protein of this invention”, as used herein, refers toany one of the new proteins isolated in accordance with this inventionand identified herein as Cry1Bf, Cry9Fa, or Cry1Jd protein. A Cryprotein, as used herein, can be a protein in the full length size, alsonamed a protoxin, or can be in a slightly or fully (e.g., N- andC-terminal truncation) truncated form as long as the insecticidalactivity is retained, or can be a combination of different proteins orprotein parts in a hybrid or fusion protein. A “Cry protoxin” refers tothe full length crystal protein as it is encoded by thenaturally-occurring Bt DNA sequence, a “Cry toxin” refers to aninsecticidal fragment thereof, particularly the smallest toxic fragmentthereof, typically in the molecular weight range of about 60 to about 80kD as determined by SDS-PAGE electrophoresis. A “cry gene” or “cry DNA”,as used herein, is a DNA sequence encoding a Cry protein in accordancewith this invention, referring to any of the cry1Bf, cry9Fa, and cry1JdDNA sequences defined above.

The “smallest toxic fragment” of a Cry protein, as used herein, is thatfragment as can be obtained by trypsin or chymotrypsin digestion of thefull length solubilized crystal protein that retains toxicity, or thattoxic protein fragment encoded by DNA fragments of the Cry protein. Thisprotein will mostly have a short N-terminal and a long C-terminaltruncation compared to the protoxin. Although for recombinantexpression, toxic fragments starting at or around original amino acidposition 1 are a more preferred embodiment in accordance with thisinvention, it should be noted that besides a C-terminal truncation, someN-terminal amino acids can also be deleted while retaining theinsecticidal character of the protein. The N-terminal end of thesmallest toxic fragment is conveniently determined by N-terminal aminoacid sequence determination of trypsin- or chymotrypsin-treated solublecrystal protein by techniques routinely available in the art.

Dna encoding the Cry proteins of this invention can be isolated in aconventional manner from the E. coli strains, deposited on Nov. 25, 1999at the BCCM-LMBP under accession numbers LMBP 3983, LMBP 3984, LMBP 3985and LMBP 3986. The encoded Cry proteins can be used to prepare specificmonoclonal or polyclonal antibodies in a conventional manner (Höfte etal., 1988). The toxin forms can be obtained by protease (e.g., trypsin)digestion of the Cry protoxins.

The DNA sequences encoding the Cry proteins can be isolated in aconventional manner from the respective strains or can be synthesizedbased on the encoded amino acid sequence.

The DNA sequences encoding the Cry proteins of the invention wereidentified by digesting total DNA from isolated Bt strains withrestriction enzymes; size fractionating the DNA fragments, so produced,into DNA fractions of 5 to 10 Kb; ligating these fractions to cloningvectors; screening the E. coli, transformed with the cloning vectors,with a DNA probe that was constructed from a region of known Bt crystalprotein genes or with a DNA probe based on specific PCR fragmentsgenerated from Bt DNA using primers corresponding to known Bt crystalprotein genes.

Also, DNA sequences for use in this invention can be made synthetically.Indeed, because of the degeneracy of the genetic code, some amino acidcodons can be replaced with others without changing the amino acidsequence of the protein. Furthermore, some amino acids can besubstituted by other equivalent amino acids without significantlychanging the insecticidal activity of the protein. Also, changes inamino acid sequence or composition in regions of the molecule, differentfrom those responsible for binding and toxicity (e.g., pore formation)are less likely to cause a difference in insecticidal activity of theprotein. Such equivalents of the gene include DNA sequences hybridizingto the DNA sequence of the Cry toxins or protoxins of SEQ ID. No. 2, 4,or 6 under stringent conditions and encoding a protein with the sameinsecticidal characteristics as the (pro)toxin of this invention, or DNAsequences encoding proteins with an amino acid sequence identity of atleast 85%, preferably at least 90%, most preferably at least 95%, withthe protein toxin form (from the N-terminus to 2 amino acids beyondconserved sequence block 5 as defined in Schnepf et al., 1998) or withthe protein protoxin form of the Cry1Bf, Cry9FA or Cry1Jd proteins ofthis invention, as determined using the GAP program of the Wisconsinpackage of GCG (Madison, Wis., USA, version 10.0; GCG defaults were usedwithin the GAP program; for the amino acid sequence comparisons, theblosum62 scoring matrix was used).

Of course, any other DNA sequence differing in its codon usage butencoding the same protein or a similar protein with substantially thesame insecticidal activity, can be constructed, depending on theparticular purpose. It has been described in prokaryotic and eucaryoticexpression systems that changing the codon usage to that of the hostcell is desired for gene expression in foreign hosts (Bennetzen & Hall,1982; Itakura, 1977). Furthermore, Bt crystal protein genes are known tohave no bias towards eucaryotic codons, and to be very AT-rich (Adang etal., 1985, Schnepf et al., 1985). Codon usage tables are available inthe literature (Wada et al., 1990; Murray et al., 1989) and in the majorDNA sequence databanks (e.g. EMBL at Heidelberg, Germany). Accordingly,synthetic DNA sequences can be constructed so that the same orsubstantially the same proteins are produced. It is evident that severalDNA sequences can be devised once the amino acid sequence of the Cryproteins of this invention is known. Such other DNA sequences includesynthetic or semi-synthetic DNA sequences that have been changed inorder to inactivate certain sites in the gene, e.g. by selectivelyinactivating certain cryptic regulatory or processing elements presentin the native sequence as described in PCT publications WO 91/16432 andWO 93/09218, or by adapting the overall codon usage to that of a morerelated host organism, preferably that of the host organism in whichexpression is desired. When making such genes, the encoded amino acidsequence should be retained to the maximum extent possible, althoughtruncations or minor replacements or additions of amino acids can bedone as long as the toxicity of the protein is not negatively affected.

Small modifications to a DNA sequence such as described above can beroutinely made by PCR-mediated mutagenesis (Ho et al., 1989, White etal., 1989).

With the term “substantially the same”, when referring to a protein, ismeant to include a protein that differs in some amino acids, or has someamino acids added (e.g. a fusion protein, see Vaeck et al., 1987) ordeleted (e.g. N- or C-terminal truncation), as long as the protein hasno major difference in its insecticidal activity.

The term “functional domain” of a Cry toxin as used herein means anypart(s) or domain(s) of the toxin with a specific structure that can betransferred to another (Cry) protein for providing a new hybrid proteinwith at least one functional characteristic (e.g., the binding and/ortoxicity characteristics) of the Cry toxin of the invention (Ge et al.,1991). Such parts can form an essential feature of the hybrid Bt proteinwith the binding and/or toxicity characteristics of the Cry protein ofthis invention. Such a hybrid protein can have an enlarged host range,an improved toxicity and/or can be used in a strategy to prevent insectresistance development (European Patent Publication (“EP”) 408 403;Visser et al., 1993).

The 5 to 10 Kb fragments, prepared from total DNA of the Bt strains ofthe invention, can be ligated in suitable expression vectors andtransformed in E. coli, and the clones can then be screened byconventional colony immunoprobing methods (French et al., 1986) forexpression of the toxin with monoclonal or polyclonal antibodies raisedagainst the Cry proteins, or by hybridization with DNA probes.

Also, the 5 to 10 Kb fragments, prepared from total DNA of the Btstrains of the invention or fragments thereof cloned and/or subcloned inE.coli, can be ligated in suitable Bt shuttle vectors (Lereclus et al.,1992) and transformed in a crystal minus Bt-mutant. The clones are thenscreened for production of crystals (detected by microscopy) or crystalproteins (detected by SDS-PAGE).

The genes encoding the Cry proteins of this invention can be sequencedin a conventional manner (Maxam and Gilbert, 1980; Sanger, 1977) toobtain the DNA sequence. Sequence comparisons indicated that the genesare different from previously described genes encoding protoxins andtoxins with activity against Lepidoptera (Höfte and Whiteley, 1989;Crickmore, et al., 1998); and the Dec. 15, 1999 and Oct. 16, 2000updates on the Bt nomenclature website corresponding to the Crickmore etal. (1998) publication, found at:

-   -   http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html

An insecticidally effective part of the DNA sequences, encoding aninsecticidally effective portion of the newly identified Cry proteinprotoxin forms, can be made in a conventional manner after sequenceanalysis of the gene. In such fragments, it is preferred that at leastthe sequence up to the C-terminal end of conserved sequence block 5 ofBt proteins (Hofte & Whiteley, 1989; Schnepf et al., 1998), preferablyup to two amino acids C-terminal of the conserved sequence block 5, isretained. The amino acid sequence of the Cry proteins can be determinedfrom the DNA sequence of the isolated DNA sequences. By “aninsecticidally effective part” of DNA sequences encoding the Cryprotein, also referred to herein as “truncated gene” or “truncated DNA”,is meant a DNA sequence encoding a polypeptide which has fewer aminoacids than the Cry protein protoxin form but which is insecticidal toinsects.

In order to express all or an insecticidally effective part of the DNAsequence encoding a Cry protein of this invention in E. coli, in otherBt strains and in plants, suitable restriction sites can be introduced,flanking the DNA sequence. This can be done by site-directedmutagenesis, using well-known procedures (Stanssens et al., 1989; Whiteet al., 1989). In order to obtain improved expression in plants, thecodon usage of the cry gene or insecticidally effective cry gene part ofthis invention can be modified to form an equivalent, modified orartificial gene or gene part in accordance with PCT publications WO91/16432 and WO 93/09218; EP 0 358 962 and EP 0 359 472, or the Bt genesor gene parts can be inserted in the chloroplast genome and expressedthere using a chloropast-active promoter (e.g., Mc Bride et al., 1995).For obtaining enhanced expression in monocot plants such as corn, amonocot intron also can be added to the chimeric gene, and the DNAsequence of the cry gene or its insecticidal part of this invention canbe further changed in a translationally neutral manner, to modifypossibly inhibiting DNA sequences present in the gene part by means ofsite-directed intron insertion and/or by introducing changes to thecodon usage, e.g., adapting the codon usage to that most preferred bythe specific plant (Murray et al., 1989) without changing significantlythe encoded amino acid sequence.

Furthermore, the binding properties of the Cry proteins of the inventioncan be evaluated, using methods known in the art (Van Rie et al., 1990),to determine if the Cry proteins of the invention bind to sites on theinsect midgut that are different from those recognized by other, knownCry or other Bt proteins. Bt toxins with different binding sites inrelevant susceptible insects are very valuable to replace known Bttoxins to which insects may have developed resistance, or to use incombination with Bt toxins having a different mode of action to preventor delay the development of insect resistance against Bt toxins,particularly when expressed in a plant. Because of the characteristicsof the newly isolated Bt toxins, they are extremely useful fortransforming plants, e.g. monocots such as corn or rice and vegetablessuch as Brassica species plants, to protect these plants from insectdamage.

The insecticidally effective cry gene part or its equivalent, preferablythe cry chimeric gene, encoding an insecticidally effective portion ofthe Cry protoxin, can be stably inserted in a conventional manner intothe nuclear genome of a single plant cell, and the so-transformed plantcell can be used in a conventional manner to produce a transformed plantthat is insect-resistant. In this regard, a disarmed Ti-plasmid,containing the insecticidally effective cry gene part, in Agrobacteriumtumefaciens can be used to transform the plant cell, and thereafter, atransformed plant can be regenerated from the transformed plant cellusing the procedures described, for example, in EP 0 116 718, EP 0 270822, PCT publication WO 84/02913 and published European Patentapplication (“EP”) 0 242 246 and in Gould et al. (1991). PreferredTi-plasmid vectors each contain the insecticidally effective cry genepart between the border sequences, or at least located to the left ofthe right border sequence, of the T-DNA of the Ti-plasmid. Of course,other types of vectors can be used to transform the plant cell, usingprocedures such as direct gene transfer (as described, for example in EP0 233 247), pollen mediated transformation (as described, for example inEP 0 270 356, PCT publication WO 85/01856, and U.S. Pat. No. 4,684,611),plant RNA virus-mediated transformation (as described, for example in EP0 067 553 and U.S. Pat. No. 4,407,956), liposome-mediated transformation(as described, for example in U.S. Pat. No. 4,536,475), and othermethods such as the recently described methods for transforming certainlines of corn (Fromm et al., 1990; Gordon-Kamm et al., 1990) and rice(Shimamoto et al., 1989; Datta et al., 1990) and the recently describedmethod for transforming monocots generally (PCT publication WO92/09696).

The resulting transformed plant can be used in a conventional plantbreeding scheme to produce more transformed plants with the samecharacteristics or to introduce the insecticidally effective cry genepart in other varieties of the same or related plant species. Seeds,which are obtained from the transformed plants, contain theinsecticidally effective cry gene part as a stable genomic insert. Cellsof the transformed plant can be cultured in a conventional manner toproduce the insecticidally effective portion of the Cry protoxin,preferably the Cry toxin, which can be recovered for use in conventionalinsecticide compositions against Lepidoptera (U.S. Pat. No. 5,254,799).In accordance with this invention, plants or seeds of the invention canbe used to obtain resistance to insects, e.g. by sowing or planting in afield wherein damaging insects usually occur, said seeds or plants.Methods for obtaining insect resistance and methods for obtainingimproved yield or reduced insect damage are thus provided in accordancewith the invention by planting or sowing in a field, preferably a fieldwherein damaging insects feeding on such plants usually occur or areexpected to occur at levels which provide economic damage to the plants,the plants of seeds of the invention producing the Cry proteins of theinvention.

The insecticidally effective cry gene part, preferably the truncated crygene, is inserted in a plant cell genome so that the inserted gene isdownstream (i.e.; 3′) of, and under the control of, a promoter which candirect the expression of the gene part in the plant cell. This ispreferably accomplished by inserting the cry chimeric gene in the plantcell genome, particularly in the nuclear or chloroplast genome.Preferred promoters include: the strong constitutive 35S promoters (the“35S promoters”) of the cauliflower mosaic virus (CaMV) of isolates CM1841 (Gardner et al., 1981), CabbB-S (Franck et al., 1980) and CabbB-JI(Hull and Howell, 1987); promoters from the ubiquitin family (e.g., themaize ubiquitin promoter of Christensen et al., 1992, see also Cornejoet al., 1993), the gos2 promoter (de Pater et al., 1992), the emupromoter (Last et al., 1990), rice actin promoters such as the promoterdescribed by Zhang et al. (1991); and the TR1′ promoter and the TR2′promoter (the “TR1′ promoter” and “TP2′ promoter”, respectively) whichdrive the expression of the 1′ and 2′ genes, respectively, of the T-DNA(Velten et al., 1984). Alternatively, a promoter can be utilized whichis not constitutive but rather is specific for one or more tissues ororgans of the plant (e.g., leaves and/or roots) whereby the inserted crygene part is expressed only in cells of the specific tissue(s) ororgan(s). For example, the insecticidally effective cry gene part couldbe selectively expressed in the leaves of a plant (e.g., corn, cotton)by placing the insecticidally effective gene part under the control of alight-inducible promoter such as the promoter of theribulose-1,5-bisphosphate carboxylase small subunit gene of the plantitself or of another plant such as pea as disclosed in U.S. Pat. No.5,254,799. Another alternative is to use a promoter whose expression isinducible (e.g., by temperature, wounding or chemical factors).

The insecticidally effective cry gene part is inserted in the plantgenome so that the inserted gene part is upstream (i.e., 5′) of suitable3′ end transcription regulation signals (i.e., transcript formation andpolyadenylation signals). This is preferably accomplished by insertingthe cry chimeric gene in the plant cell genome. Preferredpolyadenylation and transcript formation signals include those of theoctopine synthase gene (Gielen et al., 1984) and the T-DNA gene 7(Velten and Schell, 1985), which act as 3′-untranslated DNA sequences intransformed plant cells.

The insecticidally effective cry gene part can optionally be inserted inthe plant genome as a hybrid gene (U.S. Pat. No. 5,254,799; Vaeck etal., 1987) under the control of the same promoter as a selectable markergene, such as the neo gene (EP 0 242 236) encoding kanamycin resistance,so that the plant expresses a fusion protein.

All or part of the cry gene, encoding an anti-lepidopteran protein, canalso be used to transform other bacteria, such as a B. thuringiensiswhich has insecticidal activity against Lepidoptera or Coleoptera.Thereby, a transformed Bt strain can be produced which is useful forcombatting a wide spectrum of lepidopteran and coleopteran insect pestsor for combatting additional lepidopteran insect pests. Transformationof bacteria, such as bacteria of the genus Agrobacterium, Bacillus orEscherichia, with all or part of the cry gene of this invention,incorporated in a suitable cloning vehicle, can be carried out in aconventional manner, preferably using conventional electroporationtechniques as described in Mahillon et al. (1989) and in PCT Patentpublication WO 90/06999.

Transformed Bacillus species strains containing the cry gene of thisinvention can be fermented by conventional methods (Dulmage, 1981;Bernhard and Utz, 1993) to provide high yields of cells. Underappropriate conditions which are well understood (Dulmage, 1981), thesestrains each sporulate to produce crystal proteins containing the Cryprotoxin in high yields.

An insecticidal, particularly anti-lepidopteran, composition of thisinvention can be formulated in a conventional manner using themicroorganisms transformed with the cry gene, or preferably theirrespective Cry proteins or the Cry protoxin, toxin or insecticidallyeffective protoxin portion as an active ingredient, together withsuitable carriers, diluents, emulsifiers and/or dispersants (e.g., asdescribed by Bernhard and Utz, 1993). This insecticide composition canbe formulated as a wettable powder, pellets, granules or dust or as aliquid formulation with aqueous or non-aqueous solvents as a foam, gel,suspension, concentrate, etc.

A method for controlling insects, particularly Lepidoptera, inaccordance with this invention can comprise applying (e.g., spraying),to a locus (area) to be protected, an insecticidal amount of the Cryproteins or host cells transformed with the cry gene of this invention.The locus to be protected can include, for example, the habitat of theinsect pests or growing vegetation or an area where vegetation is to begrown.

To obtain the Cry protoxin or toxin, cells of the recombinant hostsexpressing the Cry protein can be grown in a conventional manner on asuitable culture medium and then lysed using conventional means such asenzymatic degradation or detergents or the like. The protoxin can thenbe separated and purified by standard techniques such as chromatography,extraction, electrophoresis, or the like. The toxin can then be obtainedby trypsin digestion of the protoxin.

The following Examples illustrate the invention, and are not provided tolimit the invention or the protection sought The sequence listingreferred to in the Examples, the Claims and the Description is asfollows:

Sequence Listing:

-   SEQ ID No. 1—amino acid and DNA sequence of Cry1Bf protein and DNA-   SEQ ID No. 2—amino acid sequence of Cry1Bf protein.-   SEQ ID No. 3—amino acid and DNA sequence of Cry1Jd protein and DNA.-   SEQ ID No. 4—amino acid sequence Cry1Jd protein.-   SEQ ID No. 5—amino acid and DNA sequence of Cry9Fa protein and DNA.-   SEQ ID No. 6—amino acid sequence of Cry9Fa protein.-   SEQ ID No. 7—DNA sequence for primer Cry1B.fw.-   SEQ ID No. 8—DNA sequence for primer B.R.-   SEQ ID No. 9—DNA sequence for primer B.F.-   SEQ ID No. 10—DNA sequence for primer JFW.-   SEQ ID No. 11—DNA sequence for primer JRV.-   SEQ ID No. 12—DNA sequence for primer 9FW.-   SEQ ID No. 13—DNA sequence for primer 9RV.    Unless otherwise stated in the Examples, all procedures for making    and manipulating recombinant DNA are carried out by the standard    procedures described in Sambrook et al., Molecular Cloning—A    Laboratory Manual, Second Ed., Cold Spring Harbor Laboratory Press,    NY (1989), and in Volumes 1 and 2 of Ausubel et al. (1994) Current    Protocols in Molecular Biology, Current Protocols, USA. Standard    materials and methods for plant molecular biology work are described    in Plant Molecular Biology Labfax (1993) by R. R. D. Croy, jointly    published by BIOS Scientific Publications Ltd (UI) and Blackwell    Scientific Publications (UI). Procedures for PCR technology can be    found in “PCR protocols: a guide to methods and applications”,    Edited by M. A. Innis, D. H. Gelfand, J. J. Sninsky and T. J. White    (Academic Press, Inc., 1990).

EXAMPLES Example 1 Characterization of the Strains

The BtS02072BG strain was isolated from a grain dust sample collected inSanto Tomas la Union, Ilocos, Philippines. The BtS02739C strain wasisolated from a grain dust sample collected in Lucena City, SouthTagalog, Philippines.

Each strain can be cultivated on conventional standard media, preferablyT₃ medium (tryptone 3 g/l, tryptose 2 g/l, yeast extract 1.5 g/l, 5 mgMnCl₂, 0.05 M Na₂HPO₄.2H₂O, 0.05 M NaH₂PO₄.H₂O, pH 6.8 and 1.5% agar),preferably at 28° C. For long term storage, it is preferred to mix anequal volume of a spore-crystal suspension with an equal volume of 50%glycerol and store this at −70° C. or lyophilize a spore-crystalsuspension. For sporulation, growth on T₃ medium is preferred for 72hours at 28° C., followed by storage at 4° C. The crystal proteinsproduced by the strains during sporulation are packaged in crystals.

Example 2 Insecticidal Activity of the BtS02072BG and BtS02739C StrainsAgainst Selected Lepidopteran Insect Species

Toxicity assays were performed on neonate larvae of Helicoverpa zea,Heliothis virescens, Ostrinia nubilalis, Spodoptera frugiperda andSesamia nonagrioides fed on an artificial diet layered withspore-crystal mixtures from either BtS02072BG or BtS02739C, at about 10⁹spore-crystals per ml.

The artificial diet (Vanderzant, 1962) was dispensed in wells of Costar24-well plates for tests on H. zea, H. virescens and O. nubilalis. 50microliter of the spore-crystal mixture was applied on the surface ofthe diet and dried in a laminar air flow. For tests on H. zea, H.virescens, one larva was placed in each well and 20 larvae were used persample. For tests on O. nubilalis, 2 larvae were placed in each well and24 larvae were used per sample. The artificial diet was dispensed inwells of Costar 48-well plates for tests on S. frugiperda and S.nonagrioides. 25 microliter of the spore-crystal mixture was applied onthe surface of the diet and dried in a laminar air flow. One larva wasplaced in each well and 18 larvae were used per sample. Dead and livinglarvae were counted on the seventh day. The percentage of dead larvaeare shown in Table I below.

TABLE I Percentage of dead larvae upon application of crystal-sporemixture to insects: BTS02072BG BTS02739C H. zea 70 15 H. virescens 85-5080-60 O. nubilalis 92 72 S. frugiperda 6 Not tested S. nonagroides 100Not tested

Example 3 Characterization of New Cry Genes

The BtS02739C genes were detected by PCR using degenerate primerstargeting conserved regions in known cry genes. The resultingamplification product was purified using the Wizard PCR preps (Promega)purification system and ligated into pGEM-T vector (Promega). Theligation mixture was electroporated into E. coli JM101. A miniprep wasmade of at least 40 insert-containing transformants, and digests wereperformed with selected restriction enzymes. Following electrophoresisof the digested miniprep DNA, different DNA fragment patterns could beobserved. For each pattern at least one colony was selected. Anappropriate DNA prep was made in order to determine the sequence of theinsert of the plasmid present in each selected colony. Alignment of thedetermined sequences of the amplification products with publiclyavailable cry sequences demonstrates that strain BtS02739C contains anovel cry1J-type gene and a novel cry9-type gene.

The BtS02072BG gene was detected as follow. First, a PCR was performedusing degenerate crystal protein gene primers on strain BtS02419J. Theresulting amplification product was used as template in a secondary PCRusing degenerate crystal protein primers

The resulting amplification product was purified using the Wizard PCRpreps (Promega) purification system and ligated into pGEM-T vector(Promega). The ligation mixture was electroporated into XL1 Blue E.coli. A miniprep was made of at least 40 insert-containingtransformants, and digests were performed with selected restrictionenzymes. Following electrophoresis of the digested miniprep DNA,different DNA fragment patterns could be observed. For each pattern atleast one colony was selected. An appropriate DNA prep was made in orderto determine the sequence of the insert of the plasmid present in eachselected colony.

From the cloned amplification products from strain BtS02419J, a sequencewas found to be identical to the corresponding fragment of cry1Be1,except for one nucleotide difference. Next, primers were selected toevaluate the presence of a cry sequence similar to that of the sequencedcry gene fragment from BtS02419J in a number of Bt strains, one of thembeing strain BtS02072BG. These primers had the following sequence (5′ to3′):

Forward primer: cry1B.fw: CAG TCC AAA CGG GTA TAA AC Reverse primer:B.R: CTG CTT CGA AGG TTG CAG TA

Alignment of the determined sequences from the amplification productswith publicly available cry sequences demonstrates that strainBtS02072BG contains a novel cry1B-type gene.

Example 4 Cloning and Expression of the Cry Genes

In order to isolate the full length cry1J-type and cry9-type gene fromBtS02739C, and the cry1B-type gene from BtS02072BG, total DNA from thesestrains was prepared and partially digested with Sau3A. The digested DNAwas size fractionated on a sucrose gradient and fragments ranging from 5Kb to 10 Kb were ligated to the BamH1-digested and TsAP (thermosensitivealkaline phosphatase)-treated cloning vector pUC19 (Yannisch-Perron etal, 1985). The ligation mixture was electroporated in E. coli XL1-Blueor E. coli JM109 cells. Transformants were plated on LB-triacillinplates containing XgaI and IPTG and white colonies were selected to beused in filter hybridization experiments. Recombinant E.coli clonescontaining the vector were then screened with the appropriate DIGlabeled probes. These probes were prepared as follows. First, a PCR wasperformed using as template cells from a recombinant E. coli clonecontaining a plasmid harboring the particular cry gene fragment,previously amplified using appropriate primers as shown in Table II.

TABLE II primers used to isolate novel Bt DNA sequences (Y = C or T, S= G or C): Length of amplified strain gene primer fragment Primersequence 2739C cry1J-type JFW 365 bp GCA GCT AAT GCT ACC ACA TC JRV GTGGCG GTA TGC TGA CTA AT cry9-type 9FW 576 GYT TTT ATT CGC CCG CCA CA 9RVCGA CAG TAG SAC CCA CTA CT 2072BG cry1B-type B.F 922 CAG CGT ATT AAG TCGATG GA B.R CTG CTT CGA AGG TTG CAG TA

The resulting amplification product was gel-purified and used astemplate in a secondary PCR reaction using DIG-labeled dNTPs. Anappropriate amount of this amplification product was used inhybridization reactions.

Colony hybridization for strain BtS02739C was performed with a mixtureof the cry1J-type probe and the cry9-type probe. Positive colonies werethen hybridized with each probe separately. Colony hybridization forstrain BtS02072BG was performed with the cry1B-type probe. Followingidentification of a positive colony containing a plasmid harboring thefull length cry gene, the sequence of the cry gene was determined usingthe dye terminator labeling method and a Perkin Elmer ABI Prism-377 DNAsequencer for both strands. Upon DNA sequencing, the genes were termedas follows: the cry1J-type and cry9-type gene from BtS02739C were namedcry1Jd and cry9Fa, respectively, and the cry1B-type gene from BtS02072BGwas named cry1Bf. The genomic sequences of the isolated cry1Jd, cry9Fa,and cry1Bf genes, as well as the proteins they encode, are shown in theSequence Listing included in this application. Comparison of thesequences with known Cry DNA or protein sequences showed that thesequences are novel and differ in a substantial number of nucleotides oramino acids from known Bt genes and proteins. Tables III-V provide anoverview of the sequence identity with respect to the coding regions ofthe most similar genes and proteins (both protoxin as toxin forms) asdetermined using the GAP program of the Wisconsin package of GCG(Madison, Wis., USA) version 10.0. GCG defaults were used within the GAPprogram. For nucleic acid sequence comparisons, the nwsgapdna scoringmatrix was used, for amino acid sequence comparisons, the blosum62scoring matrix. The toxin form, as used in Tables III-V, refers to theprotein starting at the first amino acid and ending two amino acidsbeyond the last amino acid (usually a proline) of conserved sequenceblock 5, as defined in Schnepf et al. (1998). The protoxin form refersto the entire protein or coding region of the Bt protein/gene.

TABLE III Sequence identities for cry1Bf/Cry1Bf: DNA cry1Ba1 cry1Bb1cry1Bc1 cry1Bd1 cry1Be1 Protoxin 91.912 83.890 77.207 83.565 93.774Toxin 86.562 74.922 74.922 75.342 89.220 Protein Cry1Ba1 Cry1Bb1 Cry1Bc1Cry1Bd1 Cry1Be1 Protoxin 89.869 80.193 75.795 80.933 92.170 Toxin 82.52067.868 67.868 70.142 86.499

TABLE IV Sequence identities for cry9Fa/Cry9Fa: DNA cry9Aa1 cry9Ba1cry9Ca1 cry9Da1 cry9Ea1 Protoxin 71.592 78.212 76.614 81.197 84.043Toxin 51.782 62.720 68.215 75.593 81.618 Protein Cry9Aa1 Cry9Ba1 Cry9Ca1Cry9Da1 Cry9Ea1 Protoxin 62.445 72.064 71.553 76.963 82.578 Toxin 35.82852.167 59.133 68.372 78.858

TABLE V Sequence identities for cry1Jd/Cry1Jd: DNA cry1Ja1 cry1Jb1cry1Jc1 protoxin 83.233 83.176 86.323 toxin 79.526 81.162 88.143 proteinCry1Ja1 Cry1Jb1 Cry1Jc1 protoxin 79.759 78.830 82.489 toxin 71.57474.746 81.711

Genomic clones of the newly isolated genes have been deposited at theBCCM™-LMBP (Belgian Coordinated Collections ofMicroorganisms-Laboratorium voor MoleculaireBiologie-Plasmidencollectie, University of Gent, K. L. Ledeganckstraat35, B-9000 Gent, Belgium) under the following accession numbers:

-   -   LMBP 3983 for E coli JM109 containing plasmid pUC2739C/1Jd1        comprising the cry1Jd gene, deposited on Nov. 25, 1999 (this        gene can be isolated from this plasmid on an about 8.4 kb DNA        fragment by digestion with XhoI and SmaI);    -   LMBP 3984 for E coli JM109 containing plasmid pUC2739C/9Fa1        comprising the cry9Fa gene, deposited on Nov. 25, 1999 (this        gene can be isolated from this plasmid on an about 8 kb DNA        fragment by digestion with SacI and PstI); and    -   LMBP 3986 for E coli XL1Blue containing plasmid pUC2072BG/1Bf1        comprising the cry1Bf gene, deposited on Nov. 25, 1999 (this        gene can be isolated from this plasmid on an about 7 kb DNA        fragment by digestion with SacI and SalI).

Example 5 Insecticidal Activity of the Cry Genes

The insert containing the cry9Fa gene was subcloned into a suitableshuttle vector and the resulting plasmid pSL2739C9Fa1 was introduced byroutine procedures into a crystal-minus Bt strain. The crystal proteinproduced by a sporulated culture of this recombinant Bt strain wastested on neonate larvae of H. virescens and O. nubilalis at aconcentration of about 10⁹ particles/ml. On O. nubilalis larvae, 100%mortality was observed, whereas 72% mortality was observed on H.virescens larvae, whereas after treatment with the crystal-minus controlstrain all larvae survived.

The insert containing the cry1Bf gene was subcloned into a suitableshuttle vector and the resulting plasmid pSL2072BG/1Bf was introduced byroutine procedures into a crystal-minus Bt strain. The crystal proteinproduced by a sporulated culture of this recombinant Bt strain wastested on larvae of Sesamia nonagrioides, Heliothis virescens,Helicoverpa zea and O. nubilalis at different concentrations.Significant high mortality of the Cry1Bf toxin was observed on H.virescens, Ostrinia nubilialis and Sesamia nonagrioides, while lowertoxicity was found on Helicoverpa zea. After treatment with thecrystal-minus control strain all larvae survived.

The insert containing the cry1Jd gene was subcloned into a suitableshuttle vector and the resulting plasmid pGI2739C/1Jd was introduced byroutine procedures into a crystal-minus Bt strain. The crystal proteinproduced by a sporulated culture of this recombinant Bt strain is testedon larvae of Heliothis virescens at different concentrations, andsignificant mortality of the Cry1Jd toxin was observed. After treatmentwith the crystal-minus control strain all larvae survived.

Example 6 Production of the Novel Cry Proteins in Transformed Plants

Chimeric genes encoding the truncated forms of the Cry1Bf, Cry1Jd, andCry9Fa proteins are made as described in EP 0 193 259 and published PCTpatent application WO 94/12264, using the CAMV 35S (Hull and Howell,1987) and ubiquitin (Christensen et al., 1992) promoters. Preferably,the codon usage of the open reading frame is adapted to that of the hostplant so as to optimize expression efficiency, as described in publishedPCT patent application WO 94/12264.

Rice, cotton and corn cells are transformed with the resulting chimericgenes.

Corn cells are stably transformed by either Agrobacterium-mediatedtransformation (Ishida et al., 1996, and U.S. Pat. No. 5,591,616) or byelectroporation using wounded and enzyme-degraded embryogenic callus, asdescribed in WO 92/09696 or U.S. Pat. No. 5,641,664 (incorporated hereinby reference).

Cotton cells are stably transformed by Agrobacterium-mediatedtransformation (Umbeck et al., 1987, Bio/Technology 5, 263-266; U.S.Pat. No. 5,004,863, incorporated herein by reference). Rice cells arestably transformed with the method described in published PCT patentapplication WO 92/09696.

Regenerated transformed corn, cotton and rice plants are selected byELISA, Northern and Southern blot and insecticidal effect. Chimericgene-containing progeny plants show improved resistance to insectscompared to untransformed control plants with appropriate segregation ofinsect resistance and the transformed phenotype. Protein and RNAmeasurements show that increased insect resistance is linked with higherexpression of the novel Cry protein in the plants.

REFERENCES CITED

-   Adang et al. (1985). Gene 36, 289.-   Bennetzen & Hall. (1982). J. Biol. Chem. 257, 3026-3031.-   Berhard, K L and Utz, R., “Production of Bacillus thuringiensis    insecticides for experimental and commercial uses”, In Bacillus    thuringiensis, An Environmental Biopesticide: Theory and Practice,    pp. 255-267, eds. Entwistle, P. F., Cory, J. S., Bailey, M. J. and    Higgs, S., John Wiley and Sons, New York (1993).-   Christensen et al. (1992) Plant mol. Biol. 18, 675-689.-   Cornejo et al. (1993) Plant Mol. Biol. 23, 567-581.-   Crickmore et al. (1998) Microbiol. Mol. Biol Rev. 62(3), 807-13.-   Datta S., Peterhans A., Datta K. and Potrykus I., Bio/Technology 8,    736-740 (1990).-   Dulmage, H. T., “Production of Bacteria for Biological Control of    Insects” in Biological Control in Crop Production, Ed. Paparizas,    D.C., Osmun Publishers, Totowa, N.J., USA, pp. 129-141 (1981).-   Finney, Probit Analysis, 3rd Edition, Cambridge University Press    (1971)-   Franck, Guilley, Jonard, Richards and Hirth, Cell 21, 285-294 (1980)-   French, B. T., Maul, H. N. and Maul, G. G., Anal. Biochem. 156,    417-423 (1986)-   Fromm M., Morrish F., Armstrong C., Williams R., Thomas J. and Klein    T., Bio/Technology 8, 833-839 (1990).-   Gardner, Howarth, Hahn, Brown-Luedi, Shepard and Messing, Nucleic    Acids Research 9, 2871-2887 (1981)-   Ge A., Rivers D., Milne R. and Dean D., J. Biol. Chem. 266,    17954-17958 (1991)-   Gielen, J., De Beukeleer, M., Seurinck, J., Deboeck, F., De Greve,    H., Lemmers, M., Van Montagu, M. and Schell, J., EMBO J 3, 835-845    (1984).-   Gordon-Kamm W., Spencer M., Mangano M., Adams T., Daines R., Start    W., O'Brien J., Chambers S., Adams W., Willets N., Rice T., Mackey    C., Krueger R., Kausch A. and Lemaux P., The Plant Cell 2, 603-618    (1990).-   Gould, J., Devey, M., Hasegawa, O., Ulian, E. C., Peterson, G. and    Smith, R. H., Plant Physiol. 95, 426-434 (1991).-   Ho et al. (1989). Gene 77, 51-59.-   Höfte, H., Van Rie, J., Jansens, S., Van Houtven, A., Verbruggen, H.    and Vaeck, M., Applied and Environmental Microbiology 54, 2010-2017    (1988)-   Höfte H. and Whiteley H. R., Microbiological Review 53, 242-255    (1989).-   Hull and Howell, Virology 86, 482-493 (1987)-   Ishida et al., 1996, Nature Biotechnology 14, 745-750.-   Itakura et al. (1977). Science 198, 1056-1063.-   Jansens et al. (1997) Crop Science 37, 1616-1624.-   Last et al. (1990) Theor. Appl. Genet. 81, 581-588.-   Lereclus, D.; Vallade, M.; Chaufaux, J.; Arantes, O. & Rambaud, S.,    Bio/Technology 10, 418 (1992).-   Mahillon et al, FEMS Microbiol. Letters 60, 205-210 (1989).-   Maxam, A. M. and Gilbert, W., Methods in Enzymol. 65, 499-560    (1980).-   McBride et al., 1995, Bio/Technology 13, 362-   Murray, E., Lotzer, J. and Eberle, M., Nucleic Acids Research 17(2),    477-498 (1989).-   Sanger et al., Proc. Natl. Acad. Sci. USA. 74(12), 5463-5467 (1977).-   Schnepf et al. (1985). Journal of Biological Chemistry 260, 6264.-   Schnepf et al. (1998). Microbiol. Mol. Biol. Rev. 62(3), 775-806.-   Shimamoto K, Terada R., Izawa T. and Fujimoto H., Nature 338,    274-276 (1989).-   Stanssens P., Opsomer C., McKeown Y., Kramer W., Zabeau M. and    Fritz H. J., Nucleic Acids Research 12, 4441-4454 (1989).-   Vaeck, M., Reynaerts, A., Höfte, H., Jansens, S., De Beuckeleer, M.,    Dean, C., Zabeau, M.,-   Vanderzant, J. Econ. Entomol. 55, p. 140 (1962).-   Van Montagu, M. and Leemans, J., Nature 327, 33-37(1987).-   Van Rie et al., Science 247, 72 (1990).-   Velten, J., Velten, L., Hain, R. and Schell, J., EMBO J 3, 2723-2730    (1984).-   Velten, J. and Schell, J. Nucleic Acids Research 13, 6981-6998    (1985)-   Visser, B., Bosch, D. and Honée, G., “Domain-Structure Studies of    Bacillus thuringiensis Crystal Proteins: A Genetic Approach”, In    Bacillus thuringiensis, An Environmental Biopesticide: Theory and    Practice, pp. 71-88, eds. Entwistle, P. F., Cory, J. S.,    Bailey, M. J. and Higgs, S., John Wiley and Sons, New York (1993).-   Wada et al. (1990). Nucl. Acids Res. 18, 2367-1411.-   White et al. (1989). Trends in Genet 5, 185-189.-   Yannisch-Perron, C., Vierra, J. and Messing, J., Gene 33, 103-119    (1985).-   Zhang et al. (1991) The Plant Cell 3, 1155-1165.

1. An isolated DNA encoding a protein with insecticidal activity toOstrinia nubilalis larvae, comprising an amino acid sequence at least95% identical to the amino acid sequence of SEQ ID No. 6 from amino acidposition 1 to amino acid position
 652. 2. The DNA of claim 1 encoding aprotein comprising the amino acid sequence of SEQ ID No. 6 from aminoacid position 1 to amino acid position
 652. 3. The DNA of claim 1,wherein said DNA hybridizes under stringent hybridization conditions tothe DNA of SEQ ID No. 5, wherein said stringent hybridization is by: a)immobilizing DNA on a filter, and prehybridizing said filter for 1 to 2hours in prehybridization fluid comprised of either 50% formamide, 5%SSPE, 2×Denhardt's reagent and 0.1% SDS at 42° C., or 6×SSC,2×Denhardt's reagent and 0.1% SDS at 68° C.; b) adding a denaturedlabeled probe comprising DNA of SEQ ID NO.5 directly to theprehybridization fluid and incubating for 16 to 24 hours at 42° C. ifthat was the temperature used in step a), or at 68° C. if that was thetemperature used in step a); c) after incubation, washing the filtersfor 20 minutes at room temperature in 1×SSC, 0.1% SDS, followed by threewashes of 20 minutes each at 68° C. in 0.2×SSC and 0.1% SDS; d)establishing an autoradiograph by exposing the filters for 24 to 48hours to X-ray film at −70° C. with an intensifying screen.
 4. The DNAof claim 1, comprising an artificial DNA sequence having a differentcodon usage compared to the DNA sequence of SEQ ID NO:5.
 5. An isolatedprotein encoded by the DNA of claim
 4. 6. A chimeric gene comprising theDNA of any one of claims 1 to 4 operably-linked to a plant-expressiblepromoter.
 7. A plant cell, plant or seed transformed to contain thechimeric gene of claim
 6. 8. The plant cell, plant or seed of claim 7,comprising said chimeric gene integrated in the nuclear or chloroplastDNA of their cells.
 9. The plant cell, plant or seed of claim 8, whichis selected from the group consisting of: corn, cotton, rice, oilseedrape, Brassica species, eggplant, soybean, potato, sunflower, tomato,sugarcane, tea, beans, tobacco, strawberry, clover, cucumber,watermelon, pepper, oat, barley, wheat, dahlia, gladiolus,chrysanthemum, sugarbeet, sorghum, alfalfa, and peanut.
 10. Amicro-organism, transformed to contain the DNA of any one of claims 1-4.11. The micro-organism of claim 10 which is selected from the genusAgrobacterium, Escherichia, or Bacillus.
 12. A process for controllinginsects, said process comprising expressing the DNA of any one of claims1-4 in host cells, and contacting insects with said host cells.
 13. Aprocess for obtaining a plant with resistance to insects, said processcomprising transforming plant cells with the DNA of any one of claims1-4 or with a chimeric gene comprising said DNA operably-linkled to aplant-expressible promoter, and regenerating transformed plants andprogeny thereof which are resistant to insects.
 14. The process of claim12, wherein said insects are larvae of Ostrinia nubilalis.
 15. Theprocess of claim 13, wherein said insects are larvae of Ostrinianubilalis.